According to the results of the present study, Sargassum ilicifolium and Padina australis supplementation significantly improved growth and feed efficiency in Asian seabass, Lates calcarifer. Similarly, S. ilicifolium was added to the feed of Juvenile Asian seabass at different rates (3, 6 and 9%), six weeks after feeding; all S. ilicifolium supplemented groups were improved growth performance (Zeynali et al. 2020). The improvabilities of S. ilicifolium and P. australis on the fish growth parameters may be related to their improving the gut morphology and its structure and stimulating the secretion of digestive enzymes (Zeynali et al. 2020). Previous studies have reported that inclusion of different marine seaweed meals such as Kappaphycus alvarezii (Shapawi et al. 2015), Gracilaria pygmaea (Farhoudi et al. 2020) Sargassum polycystum (Nazarudin et al. 2020) improved growth performance and/or protein efficiency ratio of Asian seabass. However, addition of Gracilaria pulvinata meal (Morshedi et al. 2018)d alvarezii, Eucheuma denticulatum and Sargassum polycystum (Shapawi and Zamry 2016) to Asian seabass feeds did not show any effect on growth performance.
The present study found that the fish fillet proximate composition was the same among all experimental groups. Similarly, many other seaweed species also not effected the fillet or whole body proximate composition of fish species, such as Dicentrarchus labrax (Peixoto et al. 2016), Oncorhynchus mykiss (Ribeiro et al. 2017), L. calcarifer (Morshedi et al. 2018; Zeynali et al. 2020), Paralichthys olivaceus (Ragaza et al. 2021), Labeo rohita (Shambhulingaiah et al. 2021) and Micropterus salmoides (Liao et al. 2022). Differently, several studies reported that the fillet or whole body proximate composition significantly changed in different fish species fed on some seaweed-incorporated diets (Ergün et al. 2009; Güroy et al. 2013; Abdel-Warith et al. 2016). These differences might be attributed to several factors such as different fish species, seaweed species, feeding period, experimental design, and specific formula of the diet or others.
Hematological parameters, white blood cells and serum biochemical parameters are a significant parameter benefitted in the evaluation of general health status in fish. The present study showed that dietary 60 g kg− 1 P. australis supplementation significantly improved the hematological profiles. This can be explained the higher bioavailability of iron for the erythropoiesis and haematosynthesis. Cian et al. (2016) reported that chelating peptides obtained from seaweed Pyropia columbina effectively enhance the bioaccessibility of iron. Earlier studies have reported that seaweed significantly enhanced the RBC counts, Hb values and/or Hct ratios of L. calcarifer (Zeynali et al. 2020), Cirrhinus mrigala (Ragunath and Ramasubramanian 2022) and Oreochromis niloticus (Nur et al. 2020; Fredrick Raja et al. 2022). In this study, an increase was observed in the granulocyte ratio and a decrease in the lymphocyte ratio was observed in fish when fed diets supplemented with P6. Similar results were recorded in S. ilicifolium (Zeynali et al. 2020) and Pangasianodon hypophthalmus (Abdelhamid et al. 2021) fed with seaweed incorporated diets. The decreasing number of circulating lymphocytes can be related to the induced transfer of lymphocytes from the blood into lymphoid organs (Yilmaz and Ergün 2018). On the other hand, the effect of seaweed to increase circulating neutrophil counts may be related to their effectiveness in inhibiting neutrophil migration through the capillary endothelial barrier by increasing TNF-α and IL-8 gene expressions, as has been reported earlier in fish fed with different feed additives (Kumari et al. 2003; Yilmaz and Ergün 2018).
In the present study, no difference occurred in the RBC count, Hb value, Hct ratio lymphocyte and neutrophil percentages in S6 group. Differently, Zeynali et al. (2020) found that S. ilicifolium meal especially at 6 and 9% significantly increased the RBC count and neutrophil ratio and decreased lymphocyte ratio of L. calcarifer compared with the control group after a 6 week feeding experiment. These different results might be associated with differences in experimental conditions and/or physiological status of fish.
In this study, serum total protein levels and WBC counts of fish fed with 6% S. ilicifolium containing feeds were the highest level whereas it did not significantly change in fish fed with 6% P. australis containing feeds. Increases in serum protein values and WBC counts are usually thought to be associated with a stronger innate immune response in fish (Yilmaz and Ergün 2018). Parallel with our study, increased serum protein values and/or WBC counts have been recorded in L. calcarifer fed with Sargassum polycystum (Nazarudin et al. 2020), L. calcarifer fed with S. ilicifolium (Zeynali et al. 2020), A. persicus fed with Gracilaria persica (Adel et al. 2021) and Sparus aurata fed with Gracilaria gracilis (Passos et al. 2021).
In our study, fish fed the S6 and P6 diets showed an increased cholesterol levels compare to fish fed on the control diet, while the incorporation of seaweed additives did not show any influence on serum triglyceride levels of experimental fish. However, in different studies, when seaweeds were added to diets of fish, it was reported that they decreased serum cholesterol or triglyceride levels of fish (Cian et al. 2019; Ale et al. 2021). Unlike our study, Zeynali et al. (2020) reported inclusion of S. ilicifolium at 3, 6 or 9% levels did not affect serum cholesterol and triglyceride in L. calcarifer. Differences between studies can be explained with the differences in feed processing, experimental condition, and physiological status of fish.
Increased serum and mucus immune parameters and/or immune related gene expression responses in L. calcarifer by seaweed additives could be due to immunomodulatory effects. No study on the effects of P. australis meal on fish immune responses has been found in the literature so far. However, P. astraulis polysaccharide extract has been reported to improve some non-specific immune parameters in shrimp (Akbary and Aminikhoei 2018; Kilawati et al. 2021).
In this study, digestive enzymes were not affected by the dietary treatments. There are contradictory data in the literature on the effect of macro algae on digestive enzymes of fish. A similar effect has also been reported in the level of total alkaline protease activity in intestinal extracts of juvenile S. aurata fed on diets supplemented with G. cornea or U. rigida (Vizcaíno et al. 2015). Moreover, seaweed supplementation (Gracilaria spp., Ulva spp. and Fucus spp.) in practical diets for European seabass and Asian seabass juveniles have no significant impact on digestive enzyme activities (Peixoto et al. 2016; Morshedi et al. 2018). In contrast with the results of this study, dietary supplementation of Ulva rigida (Akbary et al. 2018) and Padina astraulis (Hauk) (Akbary and Shahraki 2019) extracts significantly increased pancreatic digestive enzymes in grey mullet, Mugil cephalus. Moreover, the study of Zeynali et al. (2020) revealed that fish fed on the diet supplemented with 6% SIM had higher pancreatic digestive enzymes activities than other groups. The presence of soluble non starch polysaccharides (NSP) in seaweeds and the limited capacity in fish intestinal microflora to degrade NSP may reduce the rate of diffusion of digestive enzymes to substrates (Shpigel et al. 2017). In addition, the presence of several antinutrients in seaweeds may negatively affect the nutritional quality and nutrients digestion/absorption of seaweeds (Silva et al. 2015).
IGF-1 gene is mainly secreted by the liver via activation of the GH receptor. In addition, a pro-inflammatory cytokine IL1ß is produced mainly by monocytes/macrophages leading to proliferation of leucocytes, and it plays an important role in the fish immunological defenses against infections and mediates the secretion of other cytokines (Corripio-Miyar et al. 2007). Similar to our findings, it has been reported S. ilicifolium meal significantly increased serum total immunoglobulin, and liver IGF-I, IL1ß and LZ levels in L. calcarifer (Zeynali et al. 2020). Choi et al. (2014) added 0.5 and/or 1% brown macroalgae (Hizikia fusiformis) to the feeds of Paralichthys olivaceus and plasma IGF-I levels increased with increasing liver pro-inflammatory cytokine (IL-2 and IL-6). Parallel with our study, feeding L. calcarifer with 0.1 and 0.2% of Sargassum sp. extract resulted in increased total immunoglobulin and head kidney lysozyme gene expression (Yangthong et al. 2016). However, unlike our study, serum lysozyme levels decreased with increasing dietary G. pulvinata in L. calcarifer (Morshedi et al. 2018). Therefore, additional research experiments are still required to illuminate the roles of ingredients in seaweeds in the modulation of the immune response.
In conclusion, the results of the current study showed that the replacement of a small portion (6%) of dietary FM with P. australis and S. ilicifolium improved growth performance and innate immune parameters in L. calcarifer juveniles. Furthermore, whole body proximate composition and pancreatic digestive enzymes did not significantly change in different fish species fed on the seaweed-incorporated diets. At transcriptional level, our findings demonstrated that supplementing diet with P. australis and S. ilicifolium up-regulated liver IGF-1, lysozyme and IL-1β gene expression that could be resulted in higher growth rate and immunomodulatory effects of seaweeds in these groups. Overall, according to the findings of this study 6% of dietary FM could be replaced with P. australis to improve of growth and health status in L. calcarifer juveniles compared to 6% of dietary FM replaced with S. ilicifolium.